This invention provides compositions and methods relating to GBS toxin receptor polynucleotides and polypeptides. The invention relates to a receptor for a polysaccharide isolated from a bacterial source.
Group B xcex2-hemolytic Streptococci (GBS) are ubiquitous microorganisms. GBS is not known to cause any harmful infections in humans except for very young babies. GBS pneumonia, also called xe2x80x9cearly-onset diseasexe2x80x9d, is associated with high morbidity and mortality in newborn infants.
In a series of studies conducted by Dr. Carl G. Hellerqvist and his associates at the Vanderbilt University School of Medicine, Nashville, Tenn., a polysaccharide GBS toxin was identified. This toxin was determined to be a major factor in the complications of GBS pneumonia, and was found to be useful as a therapeutic agent in combating tumors though inhibition of vascularization (U.S. Pat. No. 5,010,062).
In addition, as described in U.S. Pat. No. 5,858,991 and WO98/32453, GBS toxin facilitates wound healing in patients by minimizing scarring and accelerating healing, and reduces wound-related tumor progression.
WO98/32452 and WO98/32448 describe the use of GBS toxin as a therapeutic agent for treating patients with chronic inflammatory diseases, such as rheumatoid arthritis and psoriasis, and for enhancing repair of neural injury.
Prior to this invention, receptors for GBS toxin had not been identified. The inventors, believing receptors of GBS toxin to reside on cells in the developing vasculature of tissues undergoing angiogenesis in the conditions described above, embarked upon a series of experiments resulting in the present invention.
For the first time, novel receptors for group B xcex2-hemolytic Streptococcus GBS toxin (GBS toxin receptor) have been identified. One aspect of the invention provides a polypeptide comprising a GBS toxin receptor or polypeptide fragment thereof. Preferred embodiments include mammalian GBS toxin receptors. Also provided is an antibody that recognizes GBS toxin receptor or a fragment thereof. The polypeptide of the invention can be used, inter alia, for the screening of compounds that can be used to treat or prevent conditions arising from pathologic or hypoxia-driven angiogenesis or neovascularization, such as, for example, cancerous tumors, chronic inflammatory disease, scarring during wound healing, keloids, neural injury, and reperfusion injury.
Another aspect of the invention provides a polynucleotide encoding a GBS toxin receptor or a fragment thereof and a polynucleotide hybridizable to such polynucleotide. Preferred polynucleotides are at least 10 bases in length and comprise a nucleic acid sequence encoding, or are complementary to a nucleic acid sequence encoding, a mammalian GBS toxin receptor or a polypeptide fragment thereof.
A third aspect of the invention is a complex comprising a GBS toxin bound to a mammalian toxin receptor or fragment thereof. Also provided is a method of forming such complex. The method comprises contacting a GBS toxin with a polypeptide comprising a mammalian GBS toxin receptor, or fragment thereof that can bind GBS toxin, under conditions that permit specific binding of the GBS toxin to the polypeptide, and allowing the complex to form.
Yet another aspect of the invention is a method for purifying a compound that binds a GBS toxin receptor. The method comprises providing a polypeptide comprising a mammalian GBS toxin receptor, or fragment thereof that binds GBS toxin, contacting the polypeptide with a sample comprising the compound under conditions that allow specific binding of the compound to the polypeptide, and separating the bound compound from the remainder of the sample.
Another aspect of the invention is a method of determining the presence or absence of GBS toxin in a sample. The method comprises contacting the sample with a polypeptide comprising a mammalian GBS toxin receptor, or fragment thereof that binds GBS toxin, under conditions that allow specific binding of GBS toxin to the GBS toxin receptor, and determining whether specific binding of GBS toxin has occurred. Presence of GBS toxin in a sample obtained from a neonate is indicative of early onset disease.
A sixth aspect of the invention is a method for detecting pathologic vasculature in a mammalian tissue. The method comprises detecting the presence of a GBS toxin receptor. The method can be used for detecting or monitoring a variety of medical conditions associated with angiogenesis or neovascularization, such as, for example, detecting metastasis of a cancerous tumor, or monitoring the margin of a tumor in a mammal undergoing a therapy for cancer.
Another aspect of the invention provides methods for the identification of drug candidates for the treatment of medical conditions characterized by pathologic and/or hypoxia-driven angiogenesis or neovascularization. One embodiment is a method for identifying a compound that specifically binds a mammalian GBS toxin receptor. The method comprises combining a test compound with a mammalian GBS toxin receptor, or fragment thereof that can bind GBS toxin, under conditions that allow specific binding to occur, and detecting a complex formed between the test compound and the polypeptide. Another embodiment is a method for determining cytotoxicity of a test chimeric compound. The method comprises exposing a cell expressing a mammalian GBS toxin receptor, or fragment thereof that binds GBS toxin, to a test chimeric compound comprising a cytotoxic agent coupled to GBS toxin, and detecting signs of toxicity. Yet another embodiment is a method for identifying an inhibitor of a GBS toxin receptor by incubating test cells that express GBS toxin receptor, or a fragment thereof, in the presence and absence of a test compound and under conditions in which the cells incubated in the absence of the test compound can proliferate or migrate, and comparing the proliferation or migration of the test cells incubated in the presence and absence of the test compound, wherein less proliferation or migration in the presence of the test compound is indicative of the test compound being an inhibitor of the GBS toxin receptor. An inhibitor of endothelial cell proliferation or migration can be identified by the above method, wherein less proliferation or migration of test cells in the presence of the test compound is indicative of the test compound being an inhibitor of endothelial cell proliferation or migration. A therapeutic compound for the treatment or prevention of a medical condition characterized by pathologic angiogenesis or neovascularization can also be identified by the above method, wherein less proliferation or migration of test cells in the presence of the test compound is indicative of the test compound being a candidate therapeutic compound for the treatment or prevention of the medical condition.
The invention also provides a method for identifying a compound which inhibits binding of a GBS toxin to a mammalian GBS toxin receptor. The method comprises simulating and selecting the most probable conformations of a mammalian GBS toxin receptor, designing a chemically modified analog that substantially mimics the energetically most probable three-dimensional structure of the polypeptide, chemically synthesizing the analog, and evaluating the bioactivity of the analog. Also provided is a method for identifying a compound which binds to a mammalian GBS toxin receptor. The method comprises simulating and selecting the most probable conformations of a mammalian GBS toxin receptor, deducing the most probable binding domains of the polypeptide, designing a compound that would form the energetically most probable complexes with the polypeptide, chemically synthesizing the compound, and evaluating the bioactivity of the compound.
Another aspect of the invention is a method for the prevention or treatment of neonatal onset disease in a human neonate by administering an inhibitor of binding of GBS toxin to a human GBS toxin receptor.
Yet another aspect of the invention is a method for inhibiting pathologic or hypoxia-driven endothelial cell proliferation or migration in a mammalian tissue. The method comprises specifically binding a molecule to a GBS toxin receptor present on the surface of at least one cell in the tissue, the molecule being selected from the group consisting of a compound that can evoke an inflammatory response when bound to a GBS toxin receptor in a mammal, a chimeric compound comprising a cytotoxic compound coupled to a compound that specifically binds the GBS toxin receptor, an inhibitor of GBS toxin receptor phosphorylation, and an inhibitor of GBS toxin receptor activity.
The invention also provides a GBS toxin receptor or fragment thereof, an inhibitor of a GBS toxin receptor, or an inhibitor of binding of a GBS toxin to a GBS toxin receptor, for use in a method of treatment of the human or animal body or for the manufacture of a medicament for the treatment of a medical condition characterized by pathologic or hypoxia-driven angiogenesis or neovascularization. Also provided is a chimeric compound comprising a cytotoxic agent coupled to a compound that binds GBS toxin receptor for use in a method of treatment of the human or animal body.
Also provided are pharmaceutical compositions comprising an inhibitor of a GBS toxin receptor and/or a chimeric compound comprising a cytotoxic agent coupled to a compound that binds GBS toxin receptor, and a pharmaceutically acceptable carrier.
The invention also provides kits comprising a GBS toxin receptor or fragment and/or reagents for detecting the presence of a GBS toxin receptor or polypeptide fragment thereof or the presence of a polynucleotide encoding same.